All enzymes are specific to their function. They also have a 3D shape with an active site for the catalysing of reactions, which means that each enzyme is specific for the substraite. Enzymes are proteins, and are held together by ionic bonds and strong covalent di-sulphide bonds. These bonds can be easily broken down with heat energy. This means that the protein will have changed shape or denatured, and will no longer have the active sight necessary for it to perform its original function. If it has denatured, the yeast cannot break down the glucose and will not have the necessary stuff for respiration to occur, resulting in no CO2 production.
For the investigation the source of enzymes and the organism which will carry out the respiration is yeast. The yeast will be entrapped by agar jelly. The imobilisation of the enzymes will allow them to be reused, will be stabilised more against heat or solvent effects, and will make the process continuous. Entrapment is only one method of enzyme immobilisation. This process means that the enzyme’s catalytic properties are not changed. For this investigation, agar jelly, or Alginate will be used. It is a polysaccharide from brown seaweeds and it forms a viscous solution in which not only enzymes, but whole cells can be suspended. However, it only works if the substraites are small enough so that they can diffuse through the pores of the matrix.
Preliminary Work:
During preliminary tests, I decided to look at how many yeast beads were necessary, and how much glucose solution was necessary to be used. I also wanted to see how long a sufficient amount of gas would take to be produced by counting the number of bubbles every minute for several minutes at 25oC and 40oC
Method for Experiment:
Apparatus used list:
- Bunsen Burner
- Measuring cylinder/gas syringe.
- Bungs
- Boiling Tube
- Delivery Tube
- Water Bath
- Thermometer
- 10% glucose solution
- 50 yeast beads with agar
- Stop watch
- Pipette
I will start collecting gas when the water bath has dropped to the temperature on my table. Since the rate of heat loss from the water bath was about 1oC every couple of minutes, it will not be necessary to make sure the temperature stays close to the desired temperature with the Bunsen burner. The Bunsen burner will be used at a very low strength so that the temperature increase will be gradual. The temperature will be measured with a thermometer. The delivery tube will bubble the CO2 gas through a second water bath and will be collected in a syringe filled with water. The volume of gas will be measured over five minutes. The rate will be equal to the volume divide by time, in this case 5. To test whether the gas produced is CO2, I will pass it through limewater, and if it turns cloudy, then CO2 was produced.
Diagram of apparatus:
Variables:
The key variable for this experiment will be the rate of respiration or volume of CO2 produced. The independent variable for this experiment will be the temperature of the yeast, glucose solution. The control variables will be volume of glucose solution at start, concentration of glucose solution, number of yeast beads, and the time over which CO2 is produced for each reading. The control variables are necessary because they might affect the results whereas we are only interested in the effects of temperature. To control the volume of glucose solution, a pipette and measuring cylinder will be used to measure out the exact quantity needed. The concentration at start will be at 5% because for every 95 cm3 of water, there will be 5cm3 of glucose. The number of yeast beads can be kept constant by counting them, and the duration of a reading can be measured with a stopwatch.
I will carry out 6 different readings for temp. at: 35oC, 45oC, 55oC, 65oC, 75oC and 85oC.
The range will be 50oC. I will repeat this experiment twice, so that the result can become more accurate and a trend visible in the first experiment can be confirmed by the second, and will also reveal any anomalous readings more easily.
The method I have decided to use will give accurate results and be a fair test because the temperature will be controlled accurately, and no CO2 gas will be able to escape the system. As well as this, there will be enough glucose for the yeast to respire, so there will not be a shortage of food for them. The investigation will be reliable because all of the important factors, which may affect the CO2 production rate, have been accounted for, and are being kept constant so that they do not skew the results.
Safety precautions:
As with any experiment in the laboratory, certain safety precautions must be taken:
Glassware is fragile, and may break. This might cut somebody and therefore, must be treated with care, whether unbroken or broken.
The Bunsen Burner is dangerous because it burns gas and can burn people. Since gas is quite dangerous, care must be taken to not accidentally remove the Burner from the gas mains with it still on. The water will also be heated to a high temperature, and can burn hands severely since it will be close to boiling point.
Obtaining Evidence:
Results:
Experiment 1: Experiment 2:
N.B. each volume of gas turned limewater milky to different degrees. Therefore, CO2 was produced.
The only other thing which could be worked out is whether the rate doubles with every ten degrees of temperature rise. However, this can be easily seen without any calculation.
Graphs of Results:
Analysing Evidence:
From the evidence of my results, I have found that my first hypothesis was indeed correct, and there is an increase in rate of respiration with an increase of temperature. In addition, it can be seen that the rate of respiration quickly goes down after a certain temperature, which from my scientific knowledge can be explained by the denaturation of the enzymes in the yeast. My graphs are also very similar to my prediction graph, and it seems as if there were no anomalous results. The trends from my graphs show that there is a rapid increase of rate. The rate more than doubles in both experiments at the same temperatures of 35 to 45oC and 45 to 55oC. In addition, the average rate of respiration increased by about 100%, or doubled every 10oC in both experiments. The increase was 114% (3s.f.) every 10oC. This was only the case up to the denaturation point of the majority of the enzymes.
Therefore, from my graphs several pieces of information can be either gained or extrapolated from the trend made apparent by the best fit curve confidently:
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The optimum temperature of respiration can be ascertained, about 60, 61oC
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The temperature at which the majority of the yeast cells’ enzymes have denatured, 75oC.
- There were no anomalous readings, so the procedure was good by being reliable and fair.
- The scientific knowledge about the collision theory, the kinetic theory, and the active sight of enzymes being denatured at high temperatures has been shown to be correct.
- And finally, that temperature increase beyond any doubt affects respiration rate.
All of the results therefore, can be directly attributed to the kinetic theory, the collision theory, and how enzymes are designed to break down a specific substrate with their active site. The results can all be explained because with the increase in heat energy, the kinetic energy of the molecules increases. With an increase in velocity, they move around faster which increases the probability of the substrate and the enzyme colliding. When the two collide, they react and respiration can take place because the respiration of glucose always forms carbon dioxide and energy. Therefore, simply, the higher the heat energy, the greater the rate of respiration or carbon dioxide produced. Since there is no doubt that the results are incorrect or anomalous for the glucose solution and yeast used, there is no need to redo the experiment. This is because no matter how many times an experiment is carried out, the trend will not change, but will only make it slightly more accurate, which is unnecessary. However, I was quite surprised that the yeast did manage to continue to respire at such high temperatures. Since most enzymes seem to work in nature at 40oC, 65oC seems high to be the optimum temperature. However, this can be explained by the fact that enzymes are not denatured instantly, but take some time to change. Therefore, they can take short bursts of dangerous levels of heat energy, but will soon denature.
Enzymes at a lower temperature might break down the glucose more slowly, but they will last much longer and over a long period of time, will respire just as much, if not more than the enzymes at 65oC. Therefore, as an extension of this investigation, it would be interesting to see how long it takes for the enzymes in an organism, such as yeast, to denature at different temperatures. You can then see how long it takes until respiration stops. This can then tell us the optimum temperature at which to keep enzymes for respiration in industry to get the maximum yield over a certain period of time. This would be especially useful in fermentation, so that the maximum rate of anaerobic respiration can take place over a long period of time to maximize alcohol production.
Evaluating Evidence:
The method can be seen to have been reliable and fair because there were no anomalous readings and all of the factors which could have affected or skewed the results for respiration of yeast. However, even if there were errors in the procedure, it would not make a difference since all other environmental factors were kept constant and were not changed. Therefore, if there was any error, it would have been a systematic one and constant throughout. It would not just move one point, but all of them. This means that although the values might not be correct, at least they show the correct trend. It must also be noted that the method was good because the syringe for gas collection was accurate and could be read to an accuracy of n, n.25, n.5 and n.75. This meant that values could be more exact than other syringes which could only measure to n, and n.5. As above, the results seem to be reliable because there are no anomalous ones, and all obey my prediction.
The accuracy of temperature was plus or minus 1oC, and the volume was accurate to the nearest quarter of 1cm3.
The method was suitable for this investigation because it safely and accurately found out the affect of the temperature on the rate of respiration of yeast. However, the procedure could have been better if a thermostatically controlled water bath was used, a thermometer was in the mixture of yeast and glucose solution, and if a digital manometer was used. The first would make sure that there was no temperature deviation. The second would show the real temperature at which the enzymes are working at. The third, would allow the accurate and precise collection of data for CO2 volume and it would take readings at different stages of each 5 minute period for each temperature. It could also send the data straight to a computer with software to work out the nature of the curve and certain key pieces of data. It could also be used to work out the optimum temperature more accurately.
Conclusions:
Therefore, since there are no anomalous results it is possible to make some firm conclusions about the affect of temperature on the rate of respiration of yeast:
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The optimum temperature was around 60 to 61oC.
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The majority of yeast cells had denatured at 75oC.
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On average, the rate increases by 100%, or doubles, with every extra 10oC.
- The rate of respiration falls very rapidly after the optimum temperature.
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